Molecular cloning, chromosomal localization, and cell cycle-dependent subcellular distribution of the A-kinase anchoring protein, AKAP95.
The cyclic AMP-dependent protein kinase (PKA) type II is directed to different subcellular loci through interaction of the RII subunits with A-kinase anchoring proteins (AKAPs). A full-length human clone encoding AKAP95 was identified and sequenced, and revealed a 692-amino acid open reading frame that was 89% homologous to the rat AKAP95 (V. M. Coghlan, L. K. Langeberg, A. Fernandez, N. J. Lamb, and J. D. Scott (1994) J. Biol. Chem. 269, 7658-7665). The gene encoding AKAP95 was mapped to human chromosome 19p13.1-q12 using somatic cell hybrids and PCR. A fragment covering amino acids 414-692 of human AKAP95 was expressed in Escherichia coli and shown to bind RIIalpha. Competition with a peptide covering the RII-binding domain of AKAP Ht31 abolished RIIalpha binding to AKAP95. Immunofluorescence studies in quiescent human Hs-68 fibroblasts showed a nuclear localization of AKAP95, whereas RIIalpha was excluded from the nucleus. In contrast, during mitosis AKAP95 staining was markedly changed and appeared to be excluded from the condensed chromatin and localized outside the metaphase plate. Furthermore, the subcellular localizations of AKAP95 and RIIalpha overlapped in metaphase but started to segregate in anaphase and were again separated as AKAP95 reentered the nucleus in telophase. Finally, RIIalpha was coimmunoprecipitated with AKAP95 from HeLa cells arrested in mitosis, but not from interphase HeLa cells, demonstrating a physical association between these two molecules during mitosis. The results show a distinct redistribution of AKAP95 during mitosis, suggesting that the interaction between AKAP95 and RIIalpha may be cell cycle-dependent.
Eide
T
T
Institute of Medical Biochemistry, University of Oslo, Blindern, Oslo, N-0317, Norway.
Coghlan
V
V
Orstavik
S
S
Holsve
C
C
Solberg
R
R
Skâlhegg
B S
BS
Lamb
N J
NJ
Langeberg
L
L
Fernandez
A
A
Scott
J D
JD
Jahnsen
T
T
Taskén
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D002453:Cell Cycle / Q000235:genetics*; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D002874:Chromosome Mapping; D002888:Chromosomes, Human, Pair 19 / Q000235:genetics*; D003001:Cloning, Molecular; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts; D006367:HeLa Cells; D006801:Humans; D007399:Interphase / Q000235:genetics; D047908:Intracellular Signaling Peptides and Proteins; D008938:Mitosis / Q000235:genetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D009928:Organ Specificity; D012333:RNA, Messenger / Q000032:analysis; D017386:Sequence Homology, Amino Acid; D016335:Zinc Fingers / Q000235:genetics
C490117:AKAP8L protein, human; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; C517819:PRKAR2A protein, human; C517822:Prkar2a protein, rat; D012333:RNA, Messenger; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases